Technical Field
This invention relates to new Cdk inhibitors isolated from natural sources for therapeutic uses. More particularly, it relates to compounds that are naturally occurring Cdk5 inhibitors from the plant Rhodiola rosea and their biological activities as Cdk5 inhibitors.
Background of the Invention
With today's high throughput chemical synthetic technologies and high efficiency screening methodologies, it may not be difficult to find chemical compounds that show promising biological effects at the cellular level in the laboratory. However, the rate of these compounds becoming clinically useful is very low due to a number of factors. A key factor is their toxicity and side effects, which are often severe and may not be tolerable by the human body during later stages of drug development. In this respect, new compounds discovered from natural sources that have been used therapeutically for thousands of years have received renewed attention due to the fact that they have been consumed by humans for a long time and thus, their toxicity and side effects are more likely to be more tolerable than purely synthetic compounds.
Cyclin-dependent kinases (Cdks) belong to a family of proline-directed serine/threonine kinases that play important roles in controlling cell cycle progression and transcriptional control. Activation of Cdks requires the association with specific regulatory subunits, cyclin, and requires the phosphorylation at specific threonine residues on Cdks. Cdk1, 2, 3, 4 and 6 play important roles in regulating the transition of different cell cycle phases. Cdk1 is a mitotic Cdk, whereas Cdk2, 4 and 6 are interphase Cdks that play regulatory roles in the progression of quiescent G1 to S phases. Therefore, abnormal activation of Cdks leads to cell cycle deregulation such as continued cell proliferation or unrestrained cell cycle re-entry, and subsequent development of diseases such as cancers (Shapiro, 2006; Malumbres & Barbacid, 2009). Cyclin-dependent kinase 5 (Cdk5), a proline-directed serine/threonine kinase, is unique due to its indispensable role in neuronal development and function. Despite its structural homology with other Cdks, Cdk5 may not be involved in the regulation of cell cycle. Activation of Cdk5 is dependent on its association with two specific activators, p35 and p39, which are expressed in neuronal cells. Therefore, while Cdk5 is expressed ubiquitously in cells, it is mainly active in postmitotic neurons due to the restricted expression of p35 and p39 (Tsai et al., 1993; Zheng et al., 1998).
Cdk5 plays a diverse physiological role in neural cells, including neuronal migration (Xie et al., 2003) and axon guidance (Connell-Crowley et al., 2000) during early neural development as well as synapse formation and synaptic plasticity (Cheung et al., 2006; Lai and Ip, 2009). However, more recently, Cdk5 has also been found to play important roles outside the central nervous system such as pain signaling that involves the sensory pathways (Pareek et al., 2006), and in modulating glucose-stimulated insulin levels in pancreatic beta cells, (Wei et al., 2005). Due to its key physiological roles, uncontrolled activity of Cdk5 has been linked to various diseases/disorders such that Cdk5 has emerged as a potential molecular target for therapeutic drugs. In neurons, Cdk5 deregulation triggers neuronal apoptosis (Cheung and Ip, 2004), suggesting that aberrant regulation of Cdk5 activity is responsible for the progression of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Aberrant Cdk5 activity, for example, is also linked to cancer development, progression and metastasis such as prostate and thyroid carcinoma (Strock et al., 2006; Lin et al., 2007).
The two major pathological hallmarks of AD are the accumulation of senile plaques and neurofibrillary tangles in the diseased brain. The deregulation of Cdk5 is caused by the presence of p25, a cleavage product of p35 generated under pathological conditions (Patrick et al., 1999). Accumulation of p25 protein is found in the brains of AD patients (Patrick et al., 1999). Recent findings also indicate that Cdk5 is one of the key kinases that regulate the formation of senile plaques (Monaco, 2004) and neurofibrillary tangles (Cruz et al., 2003).
Another major neurodegenerative disease that links to Cdk5 is PD). Pathologically, PD is characterized by motor impairment due to the progressive death of selected populations of neurons, especially the dopaminergic neurons in the substantia nigra pars compacta (Muntane et al., 2008). In a PD mouse model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), elevated expression and activity of Cdk5 have been reported to be correlated with dopaminergic neurons cell death (Smith et al., 2003; Qu et al., 2007). Moreover, it is of interest to note that inhibition of Cdk5 results in an increase in dopamine release, which may help ameliorate PD progression (Chergui et al., 2004). Cdk5 has also been implicated in a plethora of other neurodegenerative diseases and neurological disorders such as Huntington's disease (Anne et al., 2007), Amyotrophic Lateral Sclerosis (ALS; Bajaj et al., 1998) and ischemic injury (Wang et al., 2003).
More recently, aberrant Cdk5 activity has been linked to the pathogenesis of diabetes mellitus (type-2 diabetes). p35, the activator of Cdk5, is present in pancreatic beta cells and its activity negatively modulates insulin release in response to glucose (Wei and Tomizawa, 2007). A sustained increase in p35 protein and Cdk5 activity is reported in murine pancreatic beta cells upon high glucose exposure (Ubeda et al., 2006). Moreover, inhibition of Cdk5 activity by chemical inhibitors increases insulin secretion in cultured beta cells and in a mouse model of diabetes in a glucose-dependent manner (Ubeda et al., 2006). These findings are consistent with the observation that p35−/− mice exhibit enhanced insulin secretion upon glucose challenge (Wei et al., 2005). Cdk5 is thought to act through the regulation of the Ca2+ channel activity or regulation of insulin gene expression during glucotoxicity (Wei et al., 2005; Ubeda et al., 2006). Thus, Cdk5 inhibitors could be potential therapeutic agents for the treatment of type-2 diabetes (Kitani et al., 2007).
Cdk5 has also been emerging as a major potential target for analgesic drugs. Cdk5/p35 has been indirectly linked to nociceptive pathways. For example Cdk5 regulates the activation of mitogen activated protein kinase (MAPK) in nociceptive neurons potentially modifying the hyperalgesia that results in increased MAPK activity. Cdk5 has also been implicated in other pain pathways such as calcium calmodulin kinase II, delta FosB, the NMDA receptor and the P/Q type voltage-dependent calcium channel. Furthermore, studies suggest that Cdk5 inhibitors may be of benefit in the management of acute pain. Cdk5/p35 is shown to be involved in the processing of pain while its inhibition reduces the responsiveness of normal pain pathways (Pareek et al., 2006; Pareek and Kulkarni, 2006). More specifically, peripheral inflammation in rats induces an increase in Cdk5 activity. While p35 transgenic mice with elevated Cdk5 activity are more sensitive to painful stimuli, p35−/− and conditional Cdk5−/− mice with markedly reduced Cdk5 activity show delayed response to pain stimuli (Pareek et al., 2006; Pareek et al., 2007). Cdk5 also regulates mitogen-activated protein kinase1/2 (MEK1/2)/1M activity through a negative feedback loop during a peripheral inflammatory response (Pareek and Kulkarni, 2006). In addition, transient receptor potential vanilloid 1 (TRPV1), a ligand-gated cation channel that is activated by heat, protons and capsaicin, was recently identified as a substrate of Cdk5 (Pareek et al., 2007). Since phosphorylation of TRPV1 by Cdk5 regulates the functions of TRPV1 during pain signaling, it is believed that Cdk5 could serve as a new molecular target for developing analgesic drugs.
Since Cdk5 is associated with various diseases, screening of inhibitors that target Cdk5 may help identify potential drug leads. However, to date, only a few Cdk5 inhibitors have been identified and they are far from being ready for clinical evaluation for neuro-indications. Roscovitine, a member of the 2,6,9-substituted purine analogs, is one of the Cdk5 inhibitors in development but it also targets Cdk1, Cdk2, Cdk7 and Cdk9 (Meijer et al., 1997). Currently, roscovitine is in phase-2 clinical trials for non-small cell lung carcinoma, breast cancer, and B-cell malignancies. The indirubin family is another class of Cdk inhibitors that has its roots in Chinese medicine. The bis-indole indirubin is an active component of Ganggui Longhui Wan, a traditional Chinese medicine recipe used for the treatment of leukemia and other chronic illnesses due to its antimitotic and antitumor activities (Leclerc et al., 2001). Indirubin inhibits various kinases including Cdk1, Cdk5 as well as glycogen synthase kinase-3 beta (GSK3β). GSK3β, together with Cdk5, is believed to be responsible for the hyperphosphorylation of TAU observed in AD.
Clearly, the scientific literature strongly indicates that Cdk5 inhibitors are promising therapeutic agents for the treatment of pain and in the management of type-2 diabetes, and may also be useful in treating neurodegenerative diseases and neurological disorders. Yet, there is a lack of promising candidate compounds that can effectively inhibit Cdk5 over other Cdks.